Conjugated Polymer Process Ontology and Experimental Data Repository for Organic Field-Effect Transistors.

Polymer-based semiconductors and organic electronics encapsulate a significant research thrust for informatics-driven materials development. However, device measurements are described by a complex array of design and parameter choices, many of which are sparsely reported. For example, the mobility of a polymer-based organic field-effect transistor (OFET) may vary by several orders of magnitude for a given polymer as a plethora of parameters related to solution processing, interface design/surface treatment, thin-film deposition, postprocessing, and measurement settings have a profound effect on the value of the final measurement. Incomplete contextual, experimental details hamper the availability of reusable data applicable for data-driven optimization, modeling (e.g., machine learning), and analysis of new organic devices. To curate organic device databases that contain reproducible and findable, accessible, interoperable, and reusable (FAIR) experimental data records, data ontologies that fully describe sample provenance and process history are required. However, standards for generating such process ontologies are not widely adopted for experimental materials domains. In this work, we design and implement an object-relational database for storing experimental records of OFETs. A data structure is generated by drawing on an international standard for batch process control (ISA-88) to facilitate the design. We then mobilize these representative data records, curated from the literature and laboratory experiments, to enable data-driven learning of process-structure-property relationships. The work presented herein opens the door for the broader adoption of data management practices and design standards for both the organic electronics and the wider materials community.

Quantifying Dense Multicomponent Slurries with In-Line ATR-FTIR and Raman Spectroscopies: A Hanford Case Study.

The multiphase nature of slurries can make them difficult to process and monitor in real time. For example, the nuclear waste slurries present at the Hanford site in Washington State are multicomponent, multiphase, and inhomogeneous. Current analytical techniques for analyzing radioactive waste at Hanford rely on laboratory results from an on-site analytical laboratory, which can delay processing speed and create exposure risks for workers. However, in-line probes can provide an alternative route to collect the necessary composition information. In the present work, Raman spectroscopy and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy are tested on simulants of nuclear waste slurries containing up to 23.2 wt % solids. We observe ATR-FTIR spectroscopy to be effective in measuring the solution phase of the studied slurry systems (3.52% mean percent error), while Raman spectroscopy provides information about the suspended solids in the slurry system (18.21% mean percent error). In-line measurement of multicomponent solids typical of nuclear waste processing has been previously unreported. The composition of both the solution and solid phases is vital in ensuring stable glass formulation and effective disposal of nuclear waste at Hanford. Raman and ATR-FTIR spectroscopies can provide a safer and faster alternative for acquiring compositional information on nuclear waste slurries.

Improvement of α-amino Ester Hydrolase Stability via Computational Protein Design.

Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for ß-lactam synthesis reactions. Rational design of 'whole variants' was explored to rapidly improve AEH thermostability by mutating between 3-15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.

Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations.

The kinetics of cephalexin synthesis and hydrolysis of the activated acyl-donor precursor phenylglycine methyl ester (PGME) were characterized under a broad range of substrate concentrations. A previously developed model by Youshko-Svedas involving the formation of the acyl-enzyme complex followed by binding of the nucleophilic ß-lactam donor does not fully estimate the maximum reaction yields for cephalexin synthesis at different concentrations using initial-rate data. 7-aminodesacetoxycephalosporanic acid (7-ADCA) was discovered to be a potent inhibitor of cephalexin hydrolysis, which may account for the deviation from model predictions. Three kinetic models were compared for cephalexin synthesis, with the model incorporating competitive inhibition due to 7-ADCA yielding the best fit. Additionally, the ßF24A variant and Assemblase® did not exhibit significantly different kinetics for the synthesis of cephalexin compared to the wild-type, for the concentration range evaluated and for both initial-rate experiments and time-course synthesis experiments. Lastly, a continuous stirred-tank reactor for cephalexin synthesis was simulated using the model incorporating competitive inhibition by 7-ADCA, with clear tradeoffs observed between productivity, fractional yield, and PGME conversion.

Water-Based Dynamic Depsipeptide Chemistry: Building Block Recycling and Oligomer Distribution Control Using Hydration-Dehydration Cycles.

The high kinetic barrier to amide bond formation has historically placed narrow constraints on its utility in reversible chemistry applications. Slow kinetics has limited the use of amides for the generation of diverse combinatorial libraries and selection of target molecules. Current strategies for peptide-based dynamic chemistries require the use of nonpolar co-solvents or catalysts or the incorporation of functional groups that facilitate dynamic chemistry between peptides. In light of these limitations, we explored the use of depsipeptides: biorelevant copolymers of amino and hydroxy acids that would circumvent the challenges associated with dynamic peptide chemistry. Here, we describe a model system of N-(α-hydroxyacyl)-amino acid building blocks that reversibly polymerize to form depsipeptides when subjected to two-step evaporation-rehydration cycling under moderate conditions. The hydroxyl groups of these units allow for dynamic ester chemistry between short peptide segments through unmodified carboxyl termini. Selective recycling of building blocks is achieved by exploiting the differential hydrolytic lifetimes of depsipeptide amide and ester bonds, which we show are controllable by adjusting the solution pH, temperature, and time as well as the building blocks' side chains. We demonstrate that the polymerization and breakdown of the depsipeptides are facilitated by cyclic morpholinedione intermediates, and further show how structural properties dictate half-lives and product oligomer distributions using multifunctional building blocks. These results establish a cyclic mode of ester-based reversible depsipeptide formation that temporally separates the polymerization and depolymerization steps for the building blocks and may have implications for prebiotic polymer chemical evolution.

Reactor Design and Optimization of α-Amino Ester Hydrolase- Catalyzed Synthesis of Cephalexin.

Pharmaceutical production quality has recently been a focus for improvement through incorporation of end-to-end continuous processing. Enzymatic ß-lactam antibiotic synthesis has been one focus for continuous manufacturing, and α-amino ester hydrolases (AEHs) are currently being explored for use in the synthesis of cephalexin due to their high reactivity and selectivity. In this study, several reactors were simulated to determine how reactor type and configuration impacts reactant conversion, fractional yield toward cephalexin, and volumetric productivity for AEH-catalyzed cephalexin synthesis. The primary reactor configurations studied are single reactors including a continuous stirred-tank reactor (CSTR) and plug flow reactor (PFR) as well as two CSTRS and a CSTR + PFR in series. Substrate concentrations fed to the reactors as well as enzyme concentration in the reactor were varied. The presence of substrate inhibition was found to have a negative impact on all reactor configurations studied. No reactor configuration simultaneously allowed high substrate conversion, high fractional yield, and high productivity; however, a single PFR was found to enable the highest substrate conversion with higher fractional yields than all other reactor configurations, by minimizing substrate inhibition. Finally, to further demonstrate the impact of substrate inhibition, an AEH engineered to improve substrate inhibition was simulated and Pareto optimal fronts for a CSTR catalyzed with the current AEH were compared to Pareto fronts for the improved AEH. Overall, reduced substrate inhibition would allow for high substrate conversion, fractional yield, and productivity with only a single CSTR.

Differential Oligomerization of Alpha versus Beta Amino Acids and Hydroxy Acids in Abiotic Proto-Peptide Synthesis Reactions.

The origin of biopolymers is a central question in origins of life research. In extant life, proteins are coded linear polymers made of a fixed set of twenty alpha-L-amino acids. It is likely that the prebiotic forerunners of proteins, or protopeptides, were more heterogenous polymers with a greater diversity of building blocks and linkage stereochemistry. To investigate a possible chemical selection for alpha versus beta amino acids in abiotic polymerization reactions, we subjected mixtures of alpha and beta hydroxy and amino acids to single-step dry-down or wet-dry cycling conditions. The resulting model protopeptide mixtures were analyzed by a variety of analytical techniques, including mass spectrometry and NMR spectroscopy. We observed that amino acids typically exhibited a higher extent of polymerization in reactions that also contained alpha hydroxy acids over beta hydroxy acids, whereas the extent of polymerization by beta amino acids was higher compared to their alpha amino acid analogs. Our results suggest that a variety of heterogenous protopeptide backbones existed during the prebiotic epoch, and that selection towards alpha backbones occurred later as a result of polymer evolution.

Urea and Acetamide Rich Solutions Circumvent the Strand Inhibition Problem to Allow Multiple Rounds of DNA and RNA Copying.

For decades prebiotic chemists have attempted to achieve replication of RNA under prebiotic conditions with only limited success. One of the long-recognized impediments to achieving true replication of a duplex (copying of both strands) is the so-called strand inhibition problem. Specifically, while the two strands of an RNA (or DNA) duplex can be separated by heating, upon cooling the strands of a duplex will reanneal before mononucleotide or oligonucleotide substrates can bind to the individual strands. Here we demonstrate that a class of plausible prebiotic solvents, when coupled with thermal cycling and varying levels of hydration, circumvents the strand inhibition problem, and allows multiple rounds of information transfer from both strands of a duplex (replication). Replication was achieved by simultaneous ligation of oligomers that bind to their templates with the aid of the solvents. The solvents used consisted of concentrated solutions of urea and acetamide in water (UAcW), components that were likely abundant on the early Earth. The UAcW solvent system favors the annealing of shorter strands over the re-annealing of long strands, thereby circumventing strand inhibition. We observed an improvement of DNA and RNA replication yields by a factor of 100× over aqueous buffer. Information transfer in the UAcW solvent system is robust, being achieved for a range of solvent component ratios, various drying conditions, and in the absence or presence of added salts.

Chemical control of peptide material phase transitions.

Progressive solute-rich polymer phase transitions provide pathways for achieving ordered supramolecular assemblies. Intrinsically disordered protein domains specifically regulate information in biological networks via conformational ordering. Here we consider a molecular tagging strategy to control ordering transitions in polymeric materials and provide a proof-of-principle minimal peptide phase network captured with a dynamic chemical network.

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation*.

Chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields can range from 30 to 95 % for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3' phosphate substrates providing yields near 100 % after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.

The pH dependent mechanisms of non-enzymatic peptide bond cleavage reactions.

The non-enzymatic cleavage rates of amide bonds located in peptides in aqueous solution is pH-dependent and involves two distinct mechanisms: direct hydrolysis (herein termed "scission") and intramolecular aminolysis by the N-terminal amine (herein termed "backbiting"). While amide bond cleavage has been previously characterized using a variety of peptides, no systematic study has yet been reported addressing the effect of the pH on the interplay between the two amide bond cleavage pathways. In this study, the cleavage rates of the glycine dimer (GG), the glycine trimer (GGG), and the cyclic dimer (cGG), as well as the alanine trimer (AAA), were measured at pH 3, 5, 7, and 10 at 95 °C employing quantification based on 1H NMR. The distinct rate constants for scission and backbiting processes were obtained by solving the differential rate equations associated with the proposed kinetic model. Generalizations concerning the relative importance of the various amide bond cleavage pathways at pH 3, 5, 7, and 10 are presented. In particular, scission dominates at pH 10, while backbiting dominates at neutral pH. At the acidic pH of 3, both backbiting and scission are significant. The model of the reaction network, used in this work, enables the quantification of these multiple competing mechanisms and can be applied to longer peptides and to similar types of reaction networks.

Control of Nucleation Density in Conjugated Polymers via Seed Nucleation.

The development of processing methods to precisely control the solution state properties of semiconducting polymers in situ have remained elusive. Herein, a facile solution seed nucleation processing method is presented in which nucleated poly(3-hexylthiophene) (P3HT) solutions are blended with well-solvated, non-nucleated counterparts as a means to promote the formation of interconnected polymer networks. Nucleation and growth of these networks was induced by preprocessing the solution with UV irradiation and subsequent solution aging prior to deposition via blade-coating. This process was adopted for both batch and continuous flow processing. Superior charge carrier (hole) mobilities were observed in samples with nucleated seeds compared to controls with 0% nucleated P3HT and 100% nucleated P3HT. UV-vis spectral analysis identified that an intermediate degree of solution aggregation (15-20%) is most conducive to enhanced charge transport. The role of intrachain and interchain ordering and alignment on the mesoscale and macroscale is characterized via X-ray scattering, atomic force microscopy, and optical microscopy techniques. The results presented here provide a framework to enable in situ control of the nucleation and growth process to achieve targeted solution state properties resulting in reliable and reproducible performance when the solutions are used for device fabrication.

Understanding the Coacervate-to-Vesicle Transition of Globular Fusion Proteins to Engineer Protein Vesicle Size and Membrane Heterogeneity.

Protein-rich coacervates are liquid phases separate from the aqueous bulk phase that are used by nature for compartmentalization and more recently have been exploited by engineers for delivery and formulation applications. They also serve as an intermediate phase in an assembly path to more complex structures, such as vesicles. Recombinant fusion protein complexes made from a globular protein fused with a glutamic acid-rich leucine zipper (globule-ZE) and an arginine-rich leucine zipper fused with an elastin-like polypeptide (ZR-ELP) show different phases from soluble, through an intermediate coacervate phase, and finally to vesicles with increasing temperature of the aqueous solution. We investigated the phase transition kinetics of the fusion protein complexes at different temperatures using dynamic light scattering and microscopy, along with mathematical modeling. We controlled coacervate growth by aging the solution at an intermediate temperature that supports coacervation and confirmed that the size of the coacervate droplets dictates the size of vesicles formed upon further heating. With this understanding of the phase transition, we developed strategies to induce heterogeneity in the organization of globular proteins in the vesicle membrane through simple mixing of coacervates containing two different globular fusion proteins prior to the vesicle transition. This study gives fundamental insights and practical strategies for development of globular protein-rich coacervates and vesicles for drug delivery, microreactors, and protocell applications.

Systems Analysis for Peptide Systems Chemistry.

Living systems employ both covalent chemistry and physical assembly to achieve complex behaviors. The emerging field of systems chemistry, inspired by these biological systems, attempts to construct and analyze systems that are simpler than biology, while still embodying biological design principles. Due to the multiple phenomena at play, it can be difficult to predict which phenomena will dominate and when. Conversely, there may be no single rate-limiting step, but rather a reaction network that is difficult to intuit from a purely experimental approach. Mathematical modeling can help to sort out these issues, although it can be challenging to build such models, especially for assembly kinetics. Numerical and statistical methods can play an important role to facilitate the synergistic and iterative use of modeling and experiment, and should be part of a systems chemistry curriculum. Three case studies are presented here, from our work in peptide-based systems, to illustrate some of the tools available for model construction, model simulation, and experimental design. Examples are provided in which these tools help to evaluate hypotheses, uncover design principles, and design new experiments.

Solvent viscosity facilitates replication and ribozyme catalysis from an RNA duplex in a model prebiotic process.

The RNA World hypothesis posits that RNA was once responsible for genetic information storage and catalysis. However, a prebiotic mechanism has yet to be reported for the replication of duplex RNA that could have operated before the emergence of polymerase ribozymes. Previously, we showed that a viscous solvent enables information transfer from one strand of long RNA duplex templates, overcoming 'the strand inhibition problem'. Here, we demonstrate that the same approach allows simultaneous information transfer from both strands of long duplex templates. An additional challenge for the RNA World is that structured RNAs (like those with catalytic activity) function poorly as templates in model prebiotic RNA synthesis reactions, raising the question of how a single sequence could serve as both a catalyst and as a replication template. Here, we show that a viscous solvent also facilitates the transition of a newly synthesized hammerhead ribozyme sequence from its inactive, duplex state to its active, folded state. These results demonstrate how fluctuating environmental conditions can allow a ribozyme sequence to alternate between acting as a template for replication and functioning as a catalyst, and illustrate the potential for temporally changing environments to enable molecular processes necessary for the origin of life.

A Polymer Blend Approach for Creation of Effective Conjugated Polymer Charge Transport Pathways.

Understanding the role of the distribution of polymer chain lengths on process-structure-property relationships in semiconducting organic electronics has remained elusive due to challenges in synthesizing targeted molecular weights ( Mw) and polydispersity indices. Here, a facile blending approach of various poly(3-hexylthiophene) (P3HT) molecular weights is used to investigate the impact of the distribution of polymer chain lengths on self-assembly into aggregates and associated charge transport properties. Low and high Mw samples were blended to form a highly polydisperse sample which was compared to a similar, medium Mw control. Self-assembly was induced by preprocessing the polymer solution with UV irradiation and subsequent solution aging before deposition via blade-coating. Superior charge carrier (hole) mobilities were observed for the blend and control samples. Furthermore, their solution lifetimes exceeded 14 days. UV-vis spectral analysis suggests that low Mw P3HT lacks the mesoscale crystallinity required for percolative charge transport. In contrast, when the Mw is too high, the polymer rapidly aggregates, leading to paracrystalline disorder and structural inhomogeneity that interrupts charge-transfer pathways. The role of grain boundaries, fibrillar order, and macroscale alignment is characterized via grazing-incidence wide-angle X-ray scattering, atomic force microscopic, and optical microscopic techniques. The results presented here provide additional guidance on the interplay between polymer solubility, self-assembly, network interconnectivity, and charge transport to enable robust polymer ink formulations with reliable and reproducible performance attributes.

A Prototype of a Neural, Powered, Transtibial Prosthesis for the Cat: Benchtop Characterization.

We developed a prototype of a neural, powered, transtibial prosthesis for the use in a feline model of prosthetic gait. The prosthesis was designed for attachment to a percutaneous porous titanium implant integrated with bone, skin, and residual nerves and muscles. In the benchtop testing, the prosthesis was fixed in a testing rig and subjected to rhythmic vertical displacements and interactions with the ground at a cadence corresponding to cat walking. Several prosthesis functions were evaluated. They included sensing ground contact, control of transitions between the finite states of prosthesis loading, and a closed-loop modulation of the linear actuator gain in each loading cycle. The prosthetic design parameters (prosthesis length = 55 mm, mass = 63 g, peak extension moment = 1 Nm) corresponded closely to those of the cat foot-ankle with distal shank and the peak ankle extension moment during level walking. The linear actuator operated the prosthetic ankle joint using inputs emulating myoelectric activity of residual muscles. The linear actuator gain was modulated in each cycle to minimize the difference between the peak of ground reaction forces (GRF) recorded by a ground force sensor and a target force value. The benchtop test results demonstrated a close agreement between the GRF peaks and patterns produced by the prosthesis and by cats during level walking.

Expanding the informational chemistries of life: peptide/RNA networks.

The RNA world hypothesis simplifies the complex biopolymer networks underlining the informational and metabolic needs of living systems to a single biopolymer scaffold. This simplification requires abiotic reaction cascades for the construction of RNA, and this chemistry remains the subject of active research. Here, we explore a complementary approach involving the design of dynamic peptide networks capable of amplifying encoded chemical information and setting the stage for mutualistic associations with RNA. Peptide conformational networks are known to be capable of evolution in disease states and of co-opting metal ions, aromatic heterocycles and lipids to extend their emergent behaviours. The coexistence and association of dynamic peptide and RNA networks appear to have driven the emergence of higher-order informational systems in biology that are not available to either scaffold independently, and such mutualistic interdependence poses critical questions regarding the search for life across our Solar System and beyond.This article is part of the themed issue 'Reconceptualizing the origins of life'.

Multistep Conformation Selection in Amyloid Assembly.

Defining pathways for amyloid assembly could impact therapeutic strategies for as many as 50 disease states. Here we show that amyloid assembly is subject to different forces regulating nucleation and propagation steps and provide evidence that the more global ß-sheet/ß-sheet facial complementarity is a critical determinant for amyloid nucleation and structural selection.